Aspartase mutants and their use in r-3-aminobutyric acid synthesis

Abstract

The application provides an aspartase mutant and a method for producing R-3-aminobutyric acid by using the aspartase mutant, and belongs to the field of enzyme engineering. The amino acid sequence of the mutant is obtained by one or more times of mutation of the sequence shown in SEQ ID NO. 3. The mutant contains one or more of the four mutant sites at positions 237 (T237C), 31 (I31V), 88 (K88N) and 123 (G123S). The aspartase mutant of the application has the advantages of high catalytic activity and high selectivity. The cell obtained by fermentation can catalyze 300 g / L of substrate, which is the highest substrate concentration reported at present. When the aspartase mutant is used for synthesizing R-3-aminobutyric acid, the R-3-aminobutyric acid production rate and the chiral purity can reach more than 99% in 7 hours. Compared with the previous chemical synthesis method and biological enzyme method, the process is simple, the conversion rate is high, the production cost is relatively low, and the aspartase mutant has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering, specifically to an aspartate enzyme mutant and its application in the synthesis of R-3-aminobutyric acid. Background Technology

[0002] R-3-aminobutyric acid (CAS: 3775-73-3) is a typical chiral β-amino acid. As a precursor to the pharmaceutical intermediate R-3-aminobutanol, it can be converted to R-3-aminobutanol in a one-step reduction reaction without changing the chiral form of R-3-aminobutanol. R-3-aminobutanol (CAS: 61477-40-5) is a key intermediate in dolutegravir, a drug used to treat AIDS. Therefore, the synthesis of R-3-aminobutyric acid is of great significance for the preparation of high-purity dolutegravir.

[0003] Currently, the traditional method for producing R-3-aminobutyric acid (R-3-aminobutyric acid) is mainly chemical synthesis. However, this method is not only complex and requires stringent conditions and is difficult to operate, but also has high production costs and causes serious heavy metal pollution, hindering large-scale industrialization. In 2007, Stephen G. Davies et al. prepared R-3-aminobutyric acid using a chemical synthesis method. Using formaldehyde as a raw material, they obtained tert-butyl 2-butenoate via the Horner-Wadsworth-Emmons reaction, followed by catalytic hydrogenation to obtain tert-butyl R-3-aminobutyric acid, which was then hydrolyzed to obtain R-3-aminobutyric acid. Although this synthesis method achieved a yield of 93% and a purity of over 99%, the required conditions were extremely harsh, needing to be carried out at -78°C, making operation very difficult. CN104370755 discloses another chemical synthesis method for preparing R-3-aminobutyric acid, using ethyl acetoacetate as a raw material, condensing it with acetamide, followed by asymmetric hydrogenation and hydrolysis to obtain R-3-aminobutyric acid. This method is costly, causes serious pollution, and is detrimental to production.

[0004] In recent years, the use of enzymatic methods to produce R-3-aminobutyric acid (GABA) has become a popular trend. Taking aspartate enzyme as a catalyst, this method suffers from drawbacks such as long reaction time, low product conversion rate, and waste of reactants, often hindering large-scale industrial production. In 2014, Andreas Vogel et al. catalyzed the production of R-3-aminobutyric acid from butenoic acid using a Bacillus-derived aspartate enzyme mutant. Although this method is simple, it requires 100 hours to achieve a 60% conversion rate at a substrate concentration of 300 mM. The low substrate concentration, long reaction time, and low conversion rate make this method unsuitable for production. CN109576317A discloses another method for producing R-3-aminobutyric acid using aspartate enzyme catalysis. In addition to aspartate enzyme, the synthesis system also includes HEPES buffer and ammonium salt. While the conversion rate reaches 98%, it requires 24 hours, also exhibiting the drawback of a long reaction time.

[0005] Therefore, there is an urgent need to develop a more efficient and environmentally friendly synthetic route for R-3-aminobutyric acid. Summary of the Invention

[0006] To overcome the aforementioned deficiencies of existing technologies, the main objective of this invention is to provide a novel bio-enzymatic method for producing R-3-aminobutyric acid (GABA). Based on traditional chemical synthesis and bio-enzymatic methods, this invention employs molecular modification to design an aspartate enzyme mutant. Using this enzyme to synthesize R-3-aminobutyric acid results in a shorter reaction time and lower cost compared to previous chemical synthesis and bio-enzymatic methods, enabling large-scale industrial production of R-3-aminobutyric acid.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] (I) Selection and evolutionary screening of aspartic acid enzymes

[0009] This invention provides an aspartate enzyme mutant, which is any of the following mutants:

[0010] Mutant 4 is obtained by mutating Thr at position 237 to Cys based on the wild-type aspartic acid enzyme with the amino acid sequence shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.1;

[0011] Mutant 1, wherein mutant 1 is obtained by mutating Ile at position 31 to Val and Thr at position 237 to Cys based on wild-type aspartic acid enzyme with an amino acid sequence as shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.5;

[0012] Mutant 2, wherein mutant 2 is obtained by mutating Lys at position 88 to Asn and Thr at position 237 to Cys based on wild-type aspartic acid enzyme with amino acid sequence as shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.7;

[0013] Mutant 3 is obtained by mutating Gly at position 123 to Ser and Thr at position 237 to Cys based on the wild-type aspartic acid enzyme with the amino acid sequence shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.9.

[0014] The GenBank accession number for the aspartate enzyme encoding gene aspA is NC_000913.

[0015] The present invention also provides a nucleotide sequence encoding the above-mentioned aspartic acid enzyme mutant.

[0016] The nucleotide sequence encoding mutant 4 is shown in SEQ ID NO.2.

[0017] The nucleotide sequence encoding mutant 1 is shown in SEQ ID NO.6.

[0018] The nucleotide sequence encoding mutant 2 is shown in SEQ ID NO.8.

[0019] The nucleotide sequence encoding mutant 3 is shown in SEQ ID NO.10.

[0020] The present invention further provides a recombinant expression vector comprising the aspartate enzyme mutant encoding gene described above. Preferably, the recombinant expression vector uses pET-29a(+) as the original expression vector.

[0021] Furthermore, the present invention provides a recombinant bacterium containing the recombinant vector, preferably, the host bacterium is Escherichia coli BL21(DE3).

[0022] The present invention further provides the mutation location and method for obtaining the above-mentioned aspartate enzyme mutant. The mutation location of the aspartate enzyme mutant is described in Table 1.

[0023] Table 1. Mutation locations and methods of obtaining wild-type and mutant strains.

[0024]

[0025] This invention also provides a high-density fermentation method for exogenously expressing the above-mentioned aspartic acid enzyme mutant, comprising the following steps:

[0026] 1) Construct any of the above recombinant bacteria and culture them in a culture medium until OD200. 600 A value of 2 to 3 indicates the presence of seed solution;

[0027] 2) Take the seed culture and add it to the fermentation medium for culture. During the culture process, add carbon and nitrogen sources, and control dissolved oxygen by adjusting the aeration rate and rotation speed. Culture until OD reaches the target value. 600 With a pH of 70-90, an inducer was added to induce protein expression, resulting in a fermentation broth. The cells were collected by centrifugation, and the pH was maintained at a constant value using ammonia throughout the fermentation process.

[0028] The method for constructing the recombinant bacteria is as follows:

[0029] 1) Insert the coding gene fragment of any of the above-mentioned aspartate enzyme mutants between the NdeI and EcoRI restriction sites of the original expression vector pET-29a(+) to obtain a recombinant vector;

[0030] 2) The recombinant vector was transformed into Escherichia coli BL21(DE3) by heat shock method to obtain the recombinant bacteria.

[0031] Preferably, in step 1), the fermentation medium composition and ratio are: Na2HPO4·12H2O: 5-30 g / L, KH2PO4: 3-15 g / L, NH4Cl: 2-15 g / L, peptone: 5-40 g / L, yeast extract: 5-30 g / L, glycerol: 5-40 g / L, with water as the solvent;

[0032] Preferably, in step 2), the inducer is IPTG or lactose; more preferably, the final concentration of IPTG for induction is 0.5-1 mmol / L, and the inducer is added after 6-8 hours of fermentation to induce protein expression.

[0033] Preferably, in step 2), the pH of the fermentation process is controlled at 6.5-7.5, and the ammonia concentration is 40-60% v / v; more preferably, the ammonia concentration is 50% v / v.

[0034] Preferably, in step 2), the dissolved oxygen during fermentation is controlled at 15%-30%, and more preferably, the dissolved oxygen during fermentation is 20%. The stirring and aeration rates vary with the dissolved oxygen control, with stirring at 200-850 rpm and aeration at 1-2 vvm.

[0035] Preferably, in step 2), when the dissolved oxygen level changes abruptly, the carbon source is started to be fed in at a rate of 5-40 mL / (L·h).

[0036] The present invention also provides the application of the above-mentioned aspartic acid enzyme mutant and its recombinant strain in the synthesis of R-3-aminobutyric acid.

[0037] Specifically, under stirring conditions, the substrate is added to a conversion tank containing a certain amount of purified water, followed by the addition of a salt containing magnesium ions. Then, ammonia is used to accelerate the dissolution of the substrate and adjust the reaction pH to a fixed value. After the substrate and magnesium salt are dissolved, the temperature is controlled to the set value, and recombinant aspartate enzyme mutant bacteria are added. The volume is then made up with purified water, and the catalytic reaction begins.

[0038] After catalysis, the feed solution is passed through a ceramic membrane, and the supernatant is then passed through an ultrafiltration hollow fiber membrane. It is then decolorized with 0.5%-3% activated carbon, concentrated under reduced pressure, cooled to 2-4℃, and crystallized by adding ethanol. After centrifugation and drying, the final product is obtained.

[0039] The substrate is crotonic acid, and the magnesium salt is magnesium chloride hexahydrate.

[0040] In the synthesis system, the substrate concentration was 100-300 g / L, the magnesium ion concentration was 10-100 mmol / L, the reaction pH was 8.0-10.0, the temperature was 36-48℃, and the reaction time was 6-8 h.

[0041] Preferably, the reaction pH is 9.0 and the temperature is 42°C;

[0042] The beneficial effects of this invention are:

[0043] This invention utilizes molecular methods to obtain an aspartate enzyme mutant, which exhibits higher catalytic activity and selectivity compared to wild-type aspartate enzyme. High-density fermentation yielded cells of this mutant enzyme capable of catalyzing a substrate concentration of 300 g / L, the highest reported concentration to date. Its application in the synthesis of R-3-aminobutyric acid (GABA) allows for a single-reaction yield, achieving a GABA production rate and chiral purity exceeding 99% within 7 hours. Compared to previous chemical and enzymatic methods, this approach is not only simpler and achieves higher conversion rates but also has relatively lower production costs, demonstrating promising prospects for industrial application. Attached Figure Description

[0044] Figure 1 This is an agarose gel electrophoresis image of the successfully constructed aspartic acid mutant 4 engineered bacteria: where M is the DNA Marker and 1 is the recombinant plasmid pET29a-aspA-M4.

[0045] Figure 2 This is a growth curve of the engineered strain of aspartic acidase mutant 4 under high-density fermentation.

[0046] Figure 3 This is an SDS-PAGE electrophoresis image of the aspartate enzyme mutant 4 engineered bacteria after fermentation and induction expression: where M is the protein marker, 1 is the negative control, 2 is the sample before induction, and 3 is the sample after fermentation.

[0047] Figure 4 This is a liquid chromatography chromatogram of the synthesis of R-3-aminobutyric acid (R-3-aminobutyric acid) and precrotonic acid catalyzed by aspartate aminotransferase mutant 4.

[0048] Figure 5 This is a liquid chromatography chromatogram of the synthesis of R-3-aminobutyric acid catalyzed by aspartic acid mutant 4, which is the result of the reaction of crotonic acid and R-3-aminobutyric acid. Detailed Implementation

[0049] The following examples are provided to further illustrate the present invention, but do not limit the invention in any way. Processes and methods not described in detail in the following examples are conventional methods known in the art, and the reagents used in the examples are commercially available or prepared by methods well known to those skilled in the art. The following examples all achieve the objectives of the present invention.

[0050] Sequence Description

[0051] SEQ ID NO.1 is the amino acid sequence of the aspartic acid enzyme mutant 4aspA-M4: 478 amino acids in total, with a mutation site of T237C relative to the wild-type aspartic acid enzyme.

[0052] SEQ ID NO.2 is the nucleotide sequence of the aspartic acid enzyme mutant 4aspA-M4: 1437 bases in length.

[0053] SEQ ID NO.3 is the amino acid sequence of wild-type aspartic acid enzyme aspA-WT: 478 amino acids in total.

[0054] SEQ ID NO.4 is the nucleotide sequence of wild-type aspartic acid enzyme aspA-WT: 1437 bases in length, derived from Escherichia coli.

[0055] SEQ ID NO.5 is the amino acid sequence of the aspartic acid enzyme mutant 1aspA-M1: 478 amino acids in total, with mutation sites I31V and T237C relative to the wild-type aspartic acid enzyme.

[0056] SEQ ID NO.6 is the nucleotide sequence of the aspartic acid enzyme mutant 1aspA-M1: 1437 bases in length.

[0057] SEQ ID NO.7 is the amino acid sequence of the aspartic acid enzyme mutant 2aspA-M2: 478 amino acids in total, with mutation sites K88N and T237C relative to the wild-type aspartic acid enzyme.

[0058] SEQ ID NO.8 is the nucleotide sequence of the aspartic acid enzyme mutant 2aspA-M2: 1437 bases in length.

[0059] SEQ ID NO.9 is the amino acid sequence of the aspartic acid enzyme mutant 3aspA-M3: 478 amino acids in total, with mutation sites G123S and T237C relative to the wild-type aspartic acid enzyme.

[0060] SEQ ID NO.10 is the nucleotide sequence of the aspartic acid enzyme mutant 3aspA-M3: 1437 bases in length.

[0061] Enzyme activity definition

[0062] Under specific temperature, pH and stirring speed conditions, the amount of enzyme that converts crotonic acid to 1 μmol of R-3-aminobutyric acid per minute is called an enzyme activity unit, i.e., 1U.

[0063] Enzyme activity assay method

[0064] Add the reaction solution, which includes crotonic acid, magnesium chloride hexahydrate, ammonia, and bacterial cells, to an Erlenmeyer flask. Place the flask in a shaker for reaction, take a sample, immediately inactivate the enzyme in a metal bath, dilute, and analyze by HPLC. Calculate the enzyme activity by the change in the peak area of ​​R-3-aminobutyric acid.

[0065] Example 1: Construction of wild-type aspartic acid enzyme recombinant vector and acquisition of corresponding strain

[0066] The nucleotide sequence shown in SEQ ID NO.4 was artificially synthesized and ligated between the NdeI and EcoRI restriction sites of the pET-29a(+) expression vector to obtain the recombinant vector pET29a-aspA-WT. The recombinant vector pET29a-aspA-WT was transformed into *E. coli* BL21(DE3) and sequenced. Successful sequencing identified the strain asaspA-WT as a wild-type aspartic acid oxidase.

[0067] Example 2: Construction of recombinant vectors of mutants 1, 2, and 3 and acquisition of corresponding bacterial strains.

[0068] Random mutations were introduced using a random mutation PCR kit (catalog number BTN1010055). Using pET29a-aspA-WT as a template, the difference in R-3-aminobutyric acid (GABA) synthesis ability between the aspartate enzyme mutant and the wild type was screened under the same conditions using a activity screening method. High-activity and high-stability strains were selected, and plasmids were extracted and sent for sequencing. Based on the enzyme activity assays of nearly 100 strains, three mutants with increased activity and stability were obtained. Each mutant involved the introduction of two amino acid mutations into the wild-type amino acid sequence. The significantly altered sequences were named mutants 1-3. Mutant 1, containing the sequence at position 31 (I31V) and position 237 (T237C), was named mutant 1, and its recombinant plasmid was named pET29a-aspA-M1. Mutant 2, containing the sequence at position 88 (K88N) and position 237 (T237C), was named mutant 2, and its recombinant plasmid was named pET29a-aspA-M2. Mutant 3, containing the sequence at position 123 (G123S) and position 237 (T237C), was named mutant 3, and its recombinant plasmid was named pET29a-aspA-M3. These three recombinant vectors were transformed into E. coli BL21(DE3) and sequenced. The successfully sequenced strains were named aspA-M1, aspA-M2, and aspA-M3, respectively.

[0069] Table 2 shows the enzyme activity detection results of wild-type aspartic acid oxidase strain and three mutant strains. It can be seen that the enzyme activity of the mutant strains is significantly improved.

[0070] Table 2. Effects of amino acid sequence mutation location on enzyme activity.

[0071] strains amino acid sequence mutation location Enzyme activity (U / g) aspA-WT —— 0 aspA-M1 (I31V)(T237C) 197 aspA-M2 (K88N)(T237C) 181 aspA-M3 (G123S)(T237C) 203

[0072] Example 3: Construction of the recombinant vector of mutant 4 and acquisition of the corresponding strain

[0073] This invention does not impose any special restrictions on the method for constructing the recombinant vector; conventional methods in the art can be used. In the specific implementation of this invention, the recombinant vector is constructed using the following method: using the vector pET29a-aspA-WT as a template, site-directed mutagenesis primers Fl primer (specifically: 5'-CCTTGGTGCAACAGCAATCGGTTGTGGTCTGAACACGCCGAAAG-3') and Rl primer (specifically: 5'-CTTTCGGCGTGTTCAGACCACAACCGATTGCTGTTGCACCAAGG-3') are designed, and PCR is performed to obtain pET29a ligated into the gene T237C shown in SEQ ID NO.2; the obtained recombinant expression vector is named pET29a-aspA-M4, and the PCR reaction system is shown in Table 3, and the PCR reaction conditions are shown in Table 4.

[0074] Table 3 PCR reaction system

[0075] reaction system Volume (μL) F1 primer 1 R1 primer 1 plasmid template 1 5x PCR Buffer 10 dNTP 2.5mM 4 PrimieSTAR XGL DNA Polymerase 1 <![CDATA[dd H2O]]> 32

[0076] Table 4 PCR reaction conditions

[0077]

[0078] The PCR products were detected by gel electrophoresis, and the target band was consistent with the theoretical result, as shown in the figure. Figure 1 As shown. 20 μL of the PCR product was added to 1 μL of Dpn I restriction endonuclease to digest the template plasmid, and incubated at 37°C for 3 h. 10 μL of the digestion product was transformed into *E. coli* BL21(DE3) to obtain the corresponding recombinant *E. coli*. This recombinant *E. coli* was plated on LB agar plates containing kanamycin (100 mg / L) and cultured overnight at 37°C. Random clones were selected for colony PCR identification and sequencing verification. The results showed that the recombinant expression vector containing the gene encoding the aspartate aminotransferase mutant was successfully transformed into the expression host *E. coli* BL21(DE3), and named pET29a-mAsp. The successfully mutated bacterial culture was added to glycerol and stored at -70°C.

[0079] Example 4: High-density fermentation culture of aspartic acid enzyme mutant engineered bacteria

[0080] 1) Preparation of seed culture of aspartic acidase mutant engineered bacteria

[0081] Recombinant bacteria of aspartate aminotransferase mutant 4 were inoculated at a 10% inoculum into shake flasks containing antibiotic-containing medium and cultured at 37°C and 220 rpm for 15-16 hours until the OD reached [value missing]. 600 The value was 2-3. The culture medium used consisted of: 10 g / L tryptone, 10 g / L NaCl, and 5 g / L yeast extract.

[0082] 2) High-density fermentation of aspartic acidase mutant engineered bacteria

[0083] 5L of fermentation medium was added to a 10L fermenter and sterilized at 121℃ for 30 min. The temperature was then lowered to 37℃, the rotation speed was 200 rpm, and the aeration was 1 vvm. The seed culture from step 1) was inoculated into the fermenter at a 10% inoculum level and cultured, with dissolved oxygen controlled at 20%. After 8 h of fermentation, IPTG at a final concentration of 0.5 mmol / L was added to induce protein expression, and induction was carried out at 25℃ for 12 h. The fermentation medium composition and ratio were: Na2HPO4·12H2O 15 g / L, KH2PO4 10 g / L, NH4Cl 6 g / L, peptone 20 g / L, yeast extract 15 g / L, glycerol 15 g / L, with water as the solvent.

[0084] When the dissolved oxygen level began to change abruptly during fermentation, 50% v / v glycerol was added, with the maximum flow rate controlled at 25 mL / (L·h) throughout the fermentation process. The pH was maintained at the set value using 50% v / v ammonia throughout the fermentation process.

[0085] After 20 hours of fermentation, the OD (Oxygen Demand) was transferred to the tank. 600 The concentration was 133. The aspartate aminotransferase mutant strain obtained by centrifugation had a concentration of 192 g / L. The growth variation during high-density fermentation is shown in the graph below. Figure 2 As shown, the electrophoresis diagram after fermentation-induced expression is as follows: Figure 3 As shown.

[0086] Example 5: Synthesis of R-3-aminobutyric acid catalyzed by aspartate enzyme mutant.

[0087] Whole-cell catalytic reaction in 50 mL systems under different temperature and pH conditions

[0088] In a 50 mL system, 5 g of crotonic acid and 0.5 g of magnesium chloride hexahydrate were added. The pH was adjusted to the set value using ammonia. At the set temperature, 0.3 g of aspartic acid enzyme mutant was added with stirring at 200 rpm, and the volume was brought to 50 mL to begin the reaction. After 7 hours of reaction, the conversion rate and yield under different reaction conditions are shown in Table 5 below.

[0089] Table 5 Temperature and pH Optimization Table

[0090] Temperature / °C 36 39 42 45 48 pH 8.0 8.5 9.0 9.5 10.0 Conversion rate / % 80.2 99.3 99.8 99.1 98.2 Generation rate / % 51.9 91.4 97.8 93.1 89.5

[0091] As can be seen from the results in the table above, the enzyme exhibits the highest catalytic efficiency and best effect under the conditions of reaction temperature of 42℃ and pH of 9.0, with a conversion rate of 99.8% and a production rate of 97.8%.

[0092] Whole-cell catalytic reaction in 50 mL systems under different magnesium ion concentrations

[0093] In a 50 mL system, 5 g of crotonic acid and 0.1-1 g of magnesium chloride hexahydrate were added. The pH was adjusted to 9.0 with ammonia. At 42 °C and with stirring at 200 rpm, 0.3 g of aspartic acid enzyme mutant was added, and the volume was brought to 50 mL to begin the reaction. After 7 h of reaction, the conversion rate and yield under different reaction conditions are shown in Table 6 below.

[0094] Table 6 Optimization of Magnesium Chloride Hexahydrate Dosage

[0095] Magnesium chloride hexahydrate / g 0.1 0.3 0.5 0.7 1 Conversion rate / % 88.7 99.8 99.1 96.1 96.4 Generation rate / % 85.4 97.8 96.8 93.6 92.8

[0096] As can be seen from the results in the table above, when magnesium chloride hexahydrate is added at 0.3g, the enzyme has the highest catalytic efficiency and the best effect, with a conversion rate of 99.8% and a production rate of 97.8%.

[0097] Whole-cell catalytic reaction in 50 mL systems under different crotonic acid concentrations

[0098] In a 50 mL system, 5-15 g of crotonic acid and 0.3-0.9 g of magnesium chloride hexahydrate were added. The pH was adjusted to 9.0 with ammonia. At 42℃ and with stirring at 200 rpm, 0.3-1 g of aspartic acid enzyme mutant was added, and the volume was brought to 50 mL to initiate the reaction. After 7 hours of reaction, the conversion rate and yield under different reaction conditions are shown in Table 7 below.

[0099] Table 7 Dosage of Crotonic Acid (100-300 g / L)

[0100] Crotonic acid / g 5 10 15 Conversion rate / % 99.8 99.6 99.7 Generation rate / % 97.8 96.6 96.3

[0101] As can be seen from the results in the table above, the bacterial cells obtained by fermentation can catalyze 300 g / L of substrate, which is the highest substrate concentration reported to date, and is more conducive to industrial production.

[0102] Example 6: Synthesis of R-3-aminobutyric acid catalyzed by aspartate enzyme mutant 4 (whole-cell catalysis in 1L system)

[0103] In a 1L system, 300g of crotonic acid and 18g of magnesium chloride hexahydrate were added. The pH was adjusted to 9.0 with ammonia. 20g of recombinant bacteria (mutant 4) was added under stirring at 200rpm at 42℃, and the volume was brought to 1L to initiate the reaction. After 7 hours of reaction, the concentration of R-3-aminobutyric acid was measured to be 348g / L, with a conversion rate exceeding 99%. The liquid chromatography chromatograms of crotonic acid and R-3-aminobutyric acid at the initial and final stages of the reaction are shown below. Figure 4 , Figure 5 As shown.

[0104] After the reaction was completed, the reaction solution was passed through a ceramic membrane, and the supernatant was collected. This supernatant was then passed through an ultrafiltration hollow fiber membrane to remove proteins and other impurities, followed by decolorization with activated carbon. The decarbonized supernatant was used for concentration and crystallization, and then centrifuged and dried to obtain 317.16g of product. The product was tested and found to have a chiral purity of 99.9% and a content of 99.38%.

[0105] Example 7: Synthesis of R-3-aminobutyric acid catalyzed by aspartate enzyme mutants 1-3 (whole-cell catalysis in 1L system)

[0106] The experimental method was the same as in Example 6, except that the cells of aspartic acid mutant 4 were replaced with cells of aspartic acid mutant 1-3 respectively.

[0107] The results are shown in Table 8.

[0108] Table 8. Conversion rate, production rate, and chiral purity of R-3-aminobutyric acid synthesized by aspartic acid mutants 1-3.

[0109] R-3-aminobutyric acid Mutant 1 Mutant 2 Mutant 3 Conversion rate 92.46 93.65 94.84 Generation rate 88.43 89.85 90.08 Chiral purity 94.8 96.3 97.5

[0110] Example 8: Synthesis of R-3-aminobutyric acid catalyzed by aspartate enzyme mutant 4 (whole-cell catalysis in a 30L system)

[0111] In a 30L system, 9kg of crotonic acid and 0.54kg of magnesium chloride hexahydrate were added. The pH was adjusted to 9.0 with ammonia. 0.6kg of recombinant bacteria (mutant 4) was added at 42℃ and 200rpm with stirring. The volume was then brought to 30L to initiate the reaction. After 7 hours of reaction, the concentration of R-3-aminobutyric acid was measured to be 344.6g / L, with a conversion rate exceeding 99%.

[0112] After the reaction was completed, the reaction solution was passed through a ceramic membrane, and the supernatant was collected. This supernatant was then passed through an ultrafiltration hollow fiber membrane to remove proteins and other impurities, followed by decolorization with activated carbon. The decarbonized supernatant was used for concentration and crystallization, centrifugation, and drying to obtain 9.5 kg of product. The product was tested and found to have a chiral purity of 99.9% and a content of 99.61%.

Claims

1. An aspartate enzyme mutant, characterized in that, It is any of the following mutants: Mutant 4 is obtained by mutating Thr at position 237 to Cys based on the wild-type aspartic acid enzyme with the amino acid sequence shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.1; Mutant 1, wherein mutant 1 is obtained by mutating Ile at position 31 to Val and Thr at position 237 to Cys based on wild-type aspartic acid enzyme with an amino acid sequence as shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.5; Mutant 2, wherein mutant 2 is obtained by mutating Lys at position 88 to Asn and Thr at position 237 to Cys based on wild-type aspartic acid enzyme with amino acid sequence as shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.7; Mutant 3 is obtained by mutating Gly at position 123 to Ser and Thr at position 237 to Cys based on the wild-type aspartic acid enzyme with the amino acid sequence shown in SEQ ID NO.3, and its amino acid sequence is shown in SEQ ID NO.

9.

2. The nucleotide sequence encoding the aspartate enzyme mutant of claim 1, characterized in that, The nucleotide sequence encoding mutant 4 is shown in SEQ ID NO.2, the nucleotide sequence encoding mutant 1 is shown in SEQ ID NO.6, the nucleotide sequence encoding mutant 2 is shown in SEQ ID NO.8, and the nucleotide sequence encoding mutant 3 is shown in SEQ ID NO.

10.

3. A recombinant bacterium expressing the aspartate enzyme mutant of claim 1, characterized in that, The original expression vector of the recombinant bacteria was pET-29a(+).

4. The recombinant bacteria expressing the aspartate enzyme mutant of claim 1 according to claim 3, characterized in that, The host bacterium of the recombinant bacteria is Escherichia coli BL21 (DE3).

5. A high-density fermentation method for the aspartic acid enzyme mutant according to claim 1, characterized in that, It includes the following steps: 1) Taking the recombinant bacteria of claim 3 or 4, culturing in the medium to OD 600 value of 2~3, obtaining the seed liquid; 2) Take the seed culture, add it to the fermentation medium, and culture until OD reaches 100%. 600 With a value of 70-90, an inducer was added to induce protein expression, resulting in a fermentation broth.

6. The high-density fermentation method for the aspartic acid enzyme mutant according to claim 5, characterized in that, In step 2), the fermentation medium consists of the following components and proportions: Na2HPO4·12H2O: 5-30 g / L, KH2PO4: 3-15 g / L, NH4Cl: 2-15 g / L, peptone: 5-40 g / L, yeast extract: 5-30 g / L, glycerol: 5-40 g / L, with water as the solvent.

7. The use of the aspartic acid enzyme mutant of claim 1 in the synthesis of R-3-aminobutyric acid.

8. The use of the recombinant bacteria according to claim 3 or 4 in the synthesis of R-3-aminobutyric acid.

9. The application according to claim 8, characterized in that, In the synthesis system, the substrate is crotonic acid, with a substrate concentration of 100-300 g / L, a magnesium ion concentration of 10-100 mmol / L, a reaction pH of 8.0-10.0, a temperature of 36-48℃, and a reaction time of 6-8 h.

10. The application according to claim 9, characterized in that, In the synthesis system, the substrate concentration was 300 g / L.

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